Electrochemical biosensor for detection of DNA hydroxymethylation based on glycosylation and alkaline phosphatase catalytic signal amplification

DNA hydroxymethylation (5-hydroxymethylcytosine, 5hmC) is a kind of new epigenetic modification, which plays key roles in nuclear reprogramming, regulates the gene activity, and initiates the DNA demethylation in mammals. For further understanding the functions of 5hmC and the correlation with tumour, it is essential to develop sensitive and selective methods for detecting and sequencing 5hmC. Herein, a kind of electrochemical biosensor was fabricated for 5hmC detection based on the glycosylation modification of 5hmC and enzymatic signal amplification. Under the catalytic effect of T4 beta-glucosyltransferase, the 5hmC in target DNA was chemically modified with glucose. Then with the bridge connection of 1,4-phenyldiboronic acid, alkaline phosphatase was further captured on the electrode surface to catalyze the hydrolysis of p-nitrophenyl phosphate disodium salt to produce p-nitrophenol. Based on the relationship between the electrochemical oxidation signal of p-nitrophenol and the concentration of target DNA, the 5hmC level can be detected with high sensitivity and selectivity. The developed method also showed excellent reproducibility and stability. (C) 2015 Elsevier Ltd. All rights reserved.